What to Know About Molecular Testing and Emerging UTI Pathogens

Urinary Tract Infections (UTI) are some of the most common bacterial infections, affecting millions of people each year worldwide. Traditional urine culture (UC) has been the cornerstone of pathogen identification in UTI for over a century. However, its effectiveness and dependability have come under scrutiny due to the increase in new pathogens and complex urinary tract infections (cUTI).

Emerging pathogens are microorganisms that have recently increased in incidence, expanded their geographic or host range, or evolved new traits that enhance their ability to cause disease (Ristori et al. 2024). Emerging pathogens in the context of urinary tract infections (UTIs) are bacteria and fungi that were previously disregarded or challenging to identify using traditional techniques but are now more commonly identified as a result of developments in molecular diagnostics such as PCR. This blog discusses the main distinctions between PCR-based testing and conventional urine culture, emphasizing the ways in which molecular diagnostics are improving UTI treatment, particularly in difficult situations.

Background

Urinary Tract Infections (UTI) are some of the most common pathogenic infections in humans, with more than 400 million cases reported globally in 2019 (Yang et al. 2022, Timm et al. 2024). 

UTIs can be clinically classified into uncomplicated and complicated infections. Uncomplicated UTIs occur in patients with a healthy urinary tract system and are commonly associated with community-acquired infections. In contrast, complicated UTIs arise in patients with an impaired or obstructed urinary tract or in those using medical devices, such as catheters. Treating complicated UTIs is often more challenging due to the underlying conditions and increased risk of antibiotic-resistant pathogens (Flores-Mireles et al. 2015, Silva et al. 2022).  

UTIs are caused by a wide range of pathogens, including Gram-negative and Gram-positive bacteria, as well as fungi. Uropathogenic Escherichia coli (UPEC) bacteria are the most common pathogen, responsible for approximately 75% of all uncomplicated UTIs and 65% of complicated UTIs (Timm et al. 2024). For uncomplicated UTIs, other causative agents are (in order of prevalence) Klebsiella pneumoniae, Staphylococcus saprophyticus, Enterococcus faecalis, group B Streptococcus (GBS) including Streptococcus agalactiae, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus aureus and Candida spp. For complicated UTIs, the other causative agents are (in order of prevalence) Enterococcus spp., K. pneumoniae, Candida spp., S. aureus, P. mirabilis, P. aeruginosa and GBS (Flores-Mireles et al. 2015, Silva et al. 2022, Timm et al. 2024).

Table 1 shows the %-prevalence of UTI in uncomplicated and complicated UTI. Uncomplicated UTIs typically affect women, children and elderly patients who are otherwise healthy. Complicated UTIs are usually associated with indwelling catheters, urinary tract abnormalities, immunosuppression or exposure to antibiotics (Flores-Mireles et al. 2015, Medina et al. 2019).

Table 1 Prevalence (%) of UTI in uncomplicated and complicated UTI. Uncomplicated UTIs typically affect women, children and elderly patients who are otherwise healthy. Complicated UTIs are usually associated with indwelling catheters, urinary tract abnormalities, immunosuppression or exposure to antibiotics (Flores-Mireles et al. 2015, Medina et al. 2019).

The limitations and detection of pathogen via urine culture vs PCR

Complex urinary tract infections (cUTI) are difficult to treat, consume significant resources, and have an increased risk of causing harm to the patient (Kapoor et al. 2004). Traditional urine culture (UC) has been the cornerstone of pathogen identification in UTI for over a century, where the protocol grows microorganisms under controlled conditions from a urine sample using media. 

UC is challenged by time and technical limitations, requires both selective and non-selective media, and needs trained personnel to interpret the results (Xu et al. 2021). Conventional urine cultures (UCs) have been shown to be inadequate in diagnosing the causes of complex urinary tract infections (cUTI) (Kline et al. 2016, Kapoor et al 2024). 

Time-consuming: Urine culture requires 24-48 hours to yield results, leading to delayed diagnosis and treatment (Hao et al. 2023, Moreland et al. 2025).

Technical challenges: Urine culture is susceptible to false negatives, especially those due to anaerobic bacteria. Additionally, slow-growing uropathogens that are fastidious (i.e. any organism that has complex or particular nutritional requirements) can be difficult to culture (Xu et al. 2021). There are some benefits to an extended variant of UC that uses 5% CO₂ rather than aerobic conditions, slow-growing and/or fastidious microorganisms are still underreported (Moreland et al. 2025).

Polymicrobial infections: UC may fail to identify all pathogens, especially in polymicrobial infections (PMOs), which are common in cUTIs (Hao et al. 2023, Kapoor et al 2024)

The superiority of PCR in detecting emerging uropathogens

Polymerase Chain Reaction (PCR) pathogen detection is a molecular technique that amplifies specific DNA sequences to identify pathogens with greater sensitivity and specificity than traditional methods. Even though urine culture is the accepted method for diagnosing UTIs, PCR testing has significant benefits: 

1. PCR has a quicker turnaround time than urine culture, giving health practitioners faster results.
2. In contrast to UC, PCR tests may detect and identify infections from pathogens that are difficult to culture.

In two studies, Hao et al 2023 and Kapoor et al 2024, found that PCR was superior to UC in overall pathogen detection, and detection of both polymicrobial infections and fastidious organisms, both methods identified potentially pathogenic organisms not found in the corresponding test i.e. some pathogens were only detected in urine culture and while other pathogens only with PCR. 

As mentioned above, a fastidious organism is any organism that has complex or particular nutritional requirements, so a fastidious organism will only grow when specific nutrients are included in the medium. Hao et al. 2023, found that individual organism line-item concordance strongly favored PCR over UC for virtually all organisms on the PCR panel, with nearly half of the organisms on the PCR panel not isolated on any UC. Despite the superiority of PCR over UC, even a 45-organism PCR panel did not capture 100% of organisms isolated on UC, nor was there 100% concordance for any organism found on both tests (Hao et al 2023). Overall, the data strongly suggest that the evaluation of patients with cUTI is best accomplished using PCR in conjunction with UC (Kapoor et al 2024).

Enhanced Sensitivity: PCR can detect pathogens at low levels and identify a broader range of organisms, including those that are difficult to culture or fastidious in nature.

Faster Results: PCR tests provide quicker results, often within hours, compared to the longer wait times for urine cultures.

Better Detection of Emerging Pathogens: PCR is more effective at identifying newly emerging pathogens that may not be detected using standard culture methods.

The role of PCR for detection of emerging UTI pathogens

Moreland et al. 2023, Hao et al. 2023, Wojno et al. 2020, Kapoor et al 2024, show that new emerging pathogens for UTI such as Aerococcus urinae, Actinotignum schaalii (previously named Actinobaculum schaalii; Lotte et al 2016, Sahuquillo-Arce et al 2024) and Alloscardovia omnicolens are easily detected with PCR, but not in standard urine culture since these are fastidious organisms. Ureaplasma urealyticum and Mycoplasma hominis are two fastidious bacteria that are associated with UTI’s. They are exceptionally difficult to cultivate in standard urine culture due to their lack of cell walls (Xu et al. 2021):

A. schaalii may be missed or underdiagnosed in UC, since it requires specific growth requirements in urine culture. The natural habitat of this microorganism is not precisely known but it is probably a commensal bacterium of the urogenital tract in humans (Sahuquillo-Arce et al. 2024).

A. urinae is a rare pathogen previously thought to be a urinary contaminant lacking clinical significance, but has now received recognition as a true pathogen in UTIs. Due to similarities between aerococci and staphylococci, streptococci and enterococci these bacteria have probably been misidentified. Hence correct species determination has been difficult in the past and aerococci have been thought to be rare causes of human infection. (Rasmussen 2016, Higgins and Garg 2017).

Alloscardovia omnicolens is another emerging pathogen that can be easily detected by PCR but is often missed by conventional UC due to specific growth requirement. It is commonly thought to be a commensal for the gastrointestinal tract but has been found in clinical samples from urinary tract (Moreland et al. 2023).

Ureaplasma urealyticum is a fastidious bacterium which lacks a cell wall, and cannot be grown under standard urine culture, but can be detected by PCR. 

PCR in management of cUTI

Complex UTIs are particularly challenging to treat due to the diversity of pathogens involved and the rising issue of antibiotic resistance (Silva et al 2022). PCR testing can guide more precise treatment decisions by identifying pathogens that may not be visible through culture alone. Elia et al 2024, confirmed in their study the technical superiority of PCR over UC in overall organism detection as well as the identification of polymicrobial infections and fastidious organisms in symptomatic patients with clinical presentation of cUTI. Nearly one-third of the patients had modifications of their antibiotic regimen based on the PCR test results that would not have been found using UC alone. They found that PCR testing is a valuable tool in the management of cUTI and has utility in improving antibiotic stewardship in this difficult-to-treat subset of patient (Elia et al 2024).

The ability to identify polymicrobial infections and fastidious organisms with PCR helps clinicians select the most appropriate antibiotics, ensuring that patients receive targeted therapy rather than broad-spectrum antibiotics, which can contribute to the rise of antimicrobial resistance.

Conclusion: The Future of UTI Diagnostics

Although urine culture has been the standard method for diagnosing UTIs for over a century, its limitations are becoming more apparent, especially with the emergence of complex and polymicrobial infections. PCR-based testing offers faster, more accurate, and more comprehensive pathogen identification including detecting emerging and fastidious organisms. It has the added advantage of detecting polymicrobial infections that UC misses, making it an essential tool for managing complex UTIs.

However, PCR is not without its own limitations, and a combination of PCR and UC may provide the most complete diagnostic picture, especially for polymicrobial infections. As emerging pathogens continue to challenge traditional diagnostic methods, the integration of molecular diagnostics into clinical practice will play a key role in improving patient care, antibiotic stewardship, and the fight against antimicrobial resistance. We will in a future post discuss more details about the emerging UTI pathogens. By combining PCR with urine culture, clinicians can ensure more comprehensive pathogen detection, leading to better-targeted treatments and improved antibiotic stewardship. Antibiotic resistance (ABR) testing serves as a complementary technique to molecular diagnostics in UTI management, helping clinicians tailor antibiotic therapy based on pathogen susceptibility profiles. A more in-depth discussion on ABR testing and its role in combating resistant UTI pathogens will be explored in a future post. 

As we continue to advance our understanding of UTIs and their causes, PCR technology will undoubtedly become a cornerstone in the diagnosis and management of urinary tract infections, ensuring that patients receive timely and effective treatment.

References

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